Integrins are a family of heterodimeric class I transmembrane receptors. Individual integrins comprise an α and β subunit in non covalent association and there are known to exist at least 18 α subunits and 8 β subunits that can form 24 different heterodimers. They are involved in numerous cell-matrix and cell-cell interactions and facilitate cell adhesion, proliferation, migration and invasion. These processes occur in several normal and pathological processes, including wound healing, inflammation and tumour growth and metastasis.
αvβ6 is an epithelial cell-restricted integrin and has been shown to be expressed in malignant but not in normal epithelium. De novo expression of this integrin has been reported in oral squamous cell carcinomas (SCC) and ovarian cancer tissues and cancer cell lines. Over-expression has been reported in adenocarcinomas of the breast, and ovarian cancer, colon carcinoma, oral squamous cell carcinoma and in gastroenteropancreatic adenocarcinomas, in particular in pancreatic ductal adenocarcinomas. It has also been shown that expression of β6 in a poorly invasive SCC cell line increased migration on fibronectin and invasion through the reconstituted basement membrane, suggesting a primary role for this integrin in oral SCC invasion and metastasis. The transcriptional activation of β6 and subsequent expression of αvβ6 has been observed during the epithelial-mesenchymal transition (EMT), which allows colon carcinoma cells to acquire a more aggressive phenotype. Moreover, analysis of colorectal carcinoma samples revealed that the elevated expression is associated with a significantly reduced survival time of patients.
WO2007/039728 (Cancer Research Technology Limited) discloses experiments in which αvβ6 peptide ligands comprising the sequence motif RGDLXXL/I, wherein LXXL/I is contained within an alpha helical structure are investigated. The use of this peptide motif arose from studies in which αvβ6 expression was involved in activation of autocrine TGF-β in post-EMT cells. The Latency Associated Protein (LAP) of the LAP-TGFβ1 complex is a known ligand for αvβ6 and binding has a role in the activation of TGF-β1. The LAP protein contains the arginine-glycine-aspartic acid (RGD) sequence, a known binding motif for most integrins. In addition, a further ligand for αvβ6 is the viral protein 1 (VP1) of the foot-and-mouth disease virus (FMDV), which also contains the RGD motif. FMDV uses αvβ6 to attach to host cells and the integrin most likely also plays a role in virus uptake into endosomes. The binding of VP1 specifically to αvβ6 is mediated via residues immediately following and including the aspartic acid of the RGD motif; the DLXXL sequence has been identified as an additional αvβ6 binding motif from its ability to inhibit αvβ6-fibronectin interactions. Peptides in which either of the two leucine residues were mutated were less good as inhibitors of FMDV C-S8c1 to recognize and infect susceptible cells. The highly related RGDLXXI motif is present in the LAP protein and would be predicted to be also involved in binding with high affinity to αvβ6.
A previous study engineered a RGD motif and three RGD repeats into the CRD3 loop of an immunoglobulin human/mouse chimeric heavy chain antibody and showed that the antibody recognized specifically the integrin αvβ6(20). Similarly, a gp120 binding antibody was increased by insersting a peptide from the CD4 receptor into the CRD3 loop (21) and a DNA-binding antibody by replacing the CDR3 loop with a sequence from a class B basic helix-loop-helix protein (22). More recently peptide sequences of the prion protein that are known epitopes for monoclonal antibodies that inhibit prion disease formation were grafted into the CDR3 loop of the heavy chain of an IgG antibody specific for the envelope glycoprotein of HIV-1 (23). The resulting Prp-IgGs bound specifically to disease-associated conformations of PrP but not to the HIV envelope.